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Purpose: Testing for DNA methylation has potential in cancer screening.Most previous studies of DNA methylation in cervical cancer used a candidate gene approach.Our previous work using genome-wide DNA methylation analysis identified several novel genes that are methylated in cervical cancers.The present study was to validate the performance of these novel DNA methylation biomarkers using quantitative analysis of methylation and to compare the performance between using physician-collected and patient-collected swabs.Materials, Methods and Results: Cervical scrapings of normal cervix, low-grade lesions, high-grade lesions, invasive squamous cell carcinomas were tested using quantitative methylation specific PCR (QMSP) of SOX 1, PAX 1, LMX 1A and NKX6-1.The sensitivity of detecting cervical intraepithelial neoplasia (CIN) 3+ lesions is 100% for SOX1 and NKX6-1.The specificities of detecting CIN3+ lesions are 73.6% for SOX1 and 94.7% for NKX6-1, respectively.The accuracy ofNKX6-1 methylation for the detection of CIN3+ lesions is 99.1.There is no difference in the detection of CIN3+ lesions between using self-collected vaginal samples and physician-collected cervical scrapings.Conclusion: The application of these novel DNA methylation biomarkers in cervical cancer screening is powerful and promising.The combination of a self-sampling device and the DNA methylation detection may revolutionize the cervical cancer screening landscape in the future.